Concordance of chlamydia trachomatis infections within sexual partnerships

TECHNICAL PAPERS ONHEALTH AND BEHAVIOR MEASUREMENT

TECHNICAL PAPER 79

Concordance of chlamydia trachomatis infections within sexual partnerships

S.M. Rogers, W.C. Miller, C.F. Turner, J. Ellen, J. Zenilman, R. Rothman, M. Villarroel,A. Al-Tayyib, P. Leone, C. Gaydos, L. Ganapathi, M. Hobbs, D. Kanouse

Reference Citation

S.M. Rogers, W.C. Miller, C.F. Turner, J. Ellen, J. Zenilman, R. Rothman, M. Villarroel, A.Al-Tayyib, P. Leone, C. Gaydos, L. Ganapathi, M. Hobbs, D. Kanouse. Technical Papers onHealth and Behavior Measurement, No. 79, Washington DC: RTI Program in Health andBehavior Measurement, 2007. (Abbreviated version published in Sexually TransmittedInfections, 84: 23-28, 2008.)

Concordance of chlamydia trachomatis infectionswithin sexual partnerships

S M Rogers,1 W C Miller,2 C F Turner,1,3 J Ellen,4 J Zenilman,5 R Rothman,6

M A Villarroel,1 A Al-Tayyib,2 P Leone,2 C Gaydos,5 L Ganapathi,7 M Hobbs,2 D Kanouse8

1 Program in Health and BehaviorMeasurement, ResearchTriangle Institute, Washington,DC, USA; 2 Division of InfectiousDiseases, Department ofMedicine and Epidemiology,University of North Carolina,Chapel Hill, NC, USA; 3 CityUniversity of New York, QueensCollege and Graduate Center,New York, NY, USA; 4 Division ofAdolescent Medicine, School ofMedicine, Johns HopkinsUniversity, Baltimore, MD, USA;5 Division of Infectious Diseases,School of Medicine, JohnsHopkins University, Baltimore,MD, USA; 6 Department ofEmergency Medicine, JohnsHopkins Medical Institution,Baltimore, MD, USA; 7 ResearchComputing Division, ResearchTriangle Institute, ResearchTriangle Park, NC, USA; 8 RAND,Santa Monica, CA, USA

Correspondence to:Dr Susan M Rogers, PhD,Program in Health and BehaviorMeasurement, ResearchTriangle Institute, 701 13th StNW, Suite 750, Washington, DC20005, USA; [email protected]

Accepted 20 September 2007

ABSTRACTObjectives: The enhanced sensitivity of nucleic acidamplification tests (NAAT) provides an opportunity forestimating the prevalence of untreated Chlamydiatrachomatis infections. The transmissibility and publichealth significance of some NAAT-identified infectionsare, however, not known.Methods: Adults attending an urban emergency depart-ment provided specimens for C trachomatis screeningusing NAAT. Participants testing positive were offeredfollow-up including re-testing for C trachomatis usingNAAT and traditional methods, eg culture and directfluorescent antibody, and were treated. Partners wereoffered identical evaluation and treatment. Overall, 90 Ctrachomatis-positive participants had one or more sexualpartners enrolled.Results: Evidence of transmission, as defined by infectionconcordance between partnerships, was observed among75% of partners of index cases testing positive by bothNAAT and traditional assay but only 45% of partners ofindex cases testing positive by NAAT only (prevalenceratio 1.7, 95% CI 1.1 to 2.5). Among index participantsreturning for follow-up, 17% had no evidence of Ctrachomatis infection by NAAT or traditional assay(median follow-up three weeks).Conclusions: A substantial proportion of positive NAATresults for chlamydial infection may be of lowertransmissibility and may not persist after a short follow-up. The long-term health effects of some positive NAATare uncertain.

Unrecognised Chlamydia trachomatis infection iscommon among US young adults.1–3 Estimates ofthe prevalence of C trachomatis have been facili-tated by the development of non-invasive nucleicacid amplification tests (NAAT). NAAT providesubstantial improvements in test sensitivity whileretaining the high specificity of traditional meth-ods.4 The use of NAAT, compared with culture,increases the yield of infections detected by 20–40%.5

Although the enhanced sensitivity of NAAT iswell recognised, the significance and transmissi-bility of the additional infections detected byNAAT are unknown. It is possible that NAAT isdetecting clinically inconsequential infectionsinvolving low levels of viable organisms, or perhapsamplifiable residual DNA from a recently con-trolled infection.

We conducted a cross-sectional study withrecruitment of sexual partners to examine thepotential transmissibility of chlamydial infectionsidentified by NAAT but not by traditional assay.We tested the hypothesis that chlamydial infections

that are detectable only by NAAT are less transmis-sible, as evidenced by infection concordance withinpartnerships, than infections that are also detectableby traditional methods. As a secondary aim, weexamined the persistence of NAAT-positive infec-tions among participants after a short follow-up.

METHODSBetween November 2002 and February 2005,trained interviewers approached adult patientsattending the Johns Hopkins Hospital EmergencyDepartment to assess eligibility for C trachomatisscreening, eg age between 18 and 35 years, Englishspeaking, and sexually active in the past 90 days.Eligible adults who consented to screening alsocompleted a brief audio computer-assisted self inter-view about recent sexual and health behaviours.6

Participants screened in the emergency departmentafter March 2003 received a US$10 food coupon.

We obtained contact information from all indexparticipants undergoing C trachomatis screening inthe emergency department to facilitate the follow-up of positive test results by trained researchdisease intervention specialists. Disease interven-tion specialists offered follow-up examination todetect clinical evidence consistent with chlamydialinfection, additional C trachomatis testing (NAATand traditional assay), and treatment at the JohnsHopkins Hospital General Clinical Research Center(GCRC). Disease intervention specialists also con-tacted up to five named sexual partners within thepast 60 days and offered evaluation and treatmentprocedures identical to those provided to indexparticipants. Index participants and partners pre-senting to the GCRC for follow-up completed adetailed behavioural audio computer-assisted selfinterview and received US$50–200 in compensa-tion for their time and travel costs. Partners notattending the GCRC were offered the option of ahome visit to complete the questionnaire and tocollect a specimen for NAAT.

The Research Triangle Institute, University ofNorth Carolina, and Western (for the Johns HopkinsUniversity School of Medicine) Institutional ReviewBoards approved all study procedures. Study partici-pants with positive test results for chlamydialinfection were reported to the Baltimore CityHealth Department.

Specimen collection and laboratory testing

NAAT for C trachomatisUS Food and Drug Administration-approved NAATwere performed according to the manufacturers’instructions at the University of North Carolina.

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Initially, urine specimens were tested using a ligase chain reaction(LCR) assay (Abbott Laboratories, North Chicago, Illinois, USA).After the LCR was withdrawn from the market in 2003, maleurine specimens and female vaginal swabs were tested using theCOBAS Amplicor PCR assay (Roche Diagnostic Systems,Indianapolis, Indiana, USA).7 8 Positive NAAT results wereconfirmed by repeating the assay. Infection with C trachomatis asdetected by NAAT (N+) was defined as a repeatedly positive teston the same specimen.

At follow-up, NAAT were performed on urine (menthroughout the study and women initially), urethral swabs(men), endocervical swabs (women), and self-administeredvaginal swabs. Each index thus received three NAAT: one uponinitial recruitment in the emergency department and two repeattests on follow-up. Sexual partners received two NAAT duringtheir GCRC visit.

Traditional assay for C trachomatisTraditional testing was performed at GCRC follow-up usingculture and direct fluorescent antibody (DFA) from urethral andendocervical specimens. Specimens were stored at 280uC untiltesting by the International STD Research Laboratory, JohnsHopkins University. Culture was performed in McCoy cells.Culture-negative specimens were tested using DFA of thesediment from the centrifuged culture transport media; a slidewas considered DFA positive if three or more elementary bodieswere present. Culture-positive specimens and DFA-positive/culture-negative specimens were considered positive for Ctrachomatis by traditional assay (T+). Specimens that testedculture and DFA negative were considered traditional assaynegative (T2).

Statistical analyses and outcomesChi-square and t-tests were used to compare characteristics ofNAAT-positive index participants who did and did not presentfor follow-up.

We assessed the concordance of infection between sexualpartners as a surrogate for the transmissibility of NAAT-identified infections, restricted to partnerships in which bothNAAT and traditional test results were available. We definedconcordance as either a positive NAAT (N+) or traditional assay(T+) result for C trachomatis among partners of C trachomatis-positive index participants. All index participants were con-sidered NAAT positive on the basis of their emergencydepartment test result.

Our primary hypothesis was that chlamydial infectionsdetectable by NAAT but not by traditional assay (N+T2) inthe index participants would be less transmissible to sexualpartners than infections that were also detectable by traditionalassay (N+T+). Non-concordance was considered as evidence oflower transmissibility. We tested this hypothesis by examiningprevalence ratios with 95% CI that compared the proportion ofpartnerships with concordant C trachomatis infections (N+ and/or T+) between index participants who tested N+T2 and thosewho tested N+T+.

We also examined characteristics of index participants andtheir partners that may be associated with infection concor-dance. For all partnership analyses, we used generalisedestimating equations with a log link and binomial errordistribution to estimate prevalence ratios for concordance.Generalised estimating equations account for within-groupcorrelation, such as that that exists between multiple partnersof the same index participant.9

As a secondary aim, we examined the persistence of NAAT-identified chlamydial infections at follow-up among individualswith positive NAAT in the emergency department. Factorspotentially influencing persistence were examined in binomialregression analyses. In addition, we examined the associationbetween the persistence and concordance of infection withinpartnerships. All statistical analyses were conducted using Stataversion 8 (Stata Corp., College Station, Texas, USA).

RESULTS

Study recruitment: C trachomatis screening and follow-upOver a 27-month period, 21 trained interviewers identified 6952eligible adults attending the emergency department (fig 1); 6094(87.7%) consented to screening. The prevalence of chlamydialinfection was 7.0% in emergency department participants. Theprevalence of C trachomatis in male urine specimens (7%) wascomparable as determined by LCR and PCR (8.4% versus 6.6%,p.0.10). Among female urine specimens tested by LCR(November 2002 to August 2003), 6.8% were C trachomatispositive; 7.3% of female swab specimens tested positive by PCR(p.0.10).

Of the 419 index participants who tested NAAT positive, 81(19.3%) received antibiotic treatment during their emergencydepartment visit. Of the remaining 338, 166 (49%) participatedin follow-up. The mean number of disease interventionspecialist contacts was two (range one to 10) and the averagenumber of days between emergency department testing andfollow-up was 21.5 (range eight to 46 days). Participants atfollow-up were slightly younger (mean age 22.5 versus 23.5 years;p = 0.04) and more were women (62% versus 50.6%, p = 0.03)compared with those who did not participate (table 1).

Most index participants (87%) named one or more recentsexual partners at follow-up; 13% refused to provide partnerinformation. Of 175 partners identified, 152 (86.9%) werecontacted successfully by disease intervention specialists and102 (58.3%) attended follow-up. Nearly half (48%) of thepartnerships presented for follow-up together.

Partner concordance of C trachomatis infectionsOur concordance analyses are limited to the 83 heterosexualcouples (72 index participants with one partner, four indexeswith two partners, and one index with three partners) forwhom both NAAT and traditional assay results were available.We excluded 17 partnerships because of missing results, inade-quate specimen collection, transcription error, or multiple enroll-ment of a positive index subject. Two exclusively male partner-ships were omitted as we did not collect anal or throat specimens.

Evidence of infection transmission, as defined by concordancewithin partnerships, was more common among index casestesting positive by both NAAT and traditional assay (N+T+)than among index cases testing positive by NAAT alone(N+T2; table 2). Evidence of transmission was observed in 39of 52 partners (75%) of N+T+ index cases, but only 14 of 31partners (45%) of N+T2 index cases (prevalence ratio 1.7, 95%CI 1.1 to 2.5).

The relationship between index test result (N+T2 or N+T+)and partner concordance did not vary by gender. Among themale partners of N+T+ female indexes, 82% were concordantfor C trachomatis, compared with 46% of male partners ofN+T2 women. Similarly, 70% of female partners of N+T+index men were concordant, in comparison with 43% of femalepartners of men testing positive by NAAT only. There was nodifference by index’s or partner’s age, the number of new

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partners in the past three months, a history of chlamydialinfection, a history of any sexually transmitted infection,antibiotic use before the follow-up visit, or time to indexfollow-up. Adjusting for the timing of the partner visit,however, reduced the prevalence ratio to 1.5 (95% CI 1.03 to2.2). Partners presenting at the same time as the indexparticipant were more likely to test concordant than partnerspresenting after the index visit.

Limiting traditional test results to culture only decreased ouroverall estimate of concordance from 64% to 54% (prevalenceratio 1.4, 95% CI 0.93 to 2.0). Differences in the type of NAAT(LCR versus PCR) or specimen type (female urine versus vaginalswab) could also influence our transmission estimates.Although all women screened in the emergency departmentprovided vaginal swabs for C trachomatis testing using PCR,initially women also provided urine specimens for LCR testing.When the LCR was withdrawn in 2003, male urine samples andfemale swabs were tested using PCR. Restricting our con-cordance analysis to include only female indexes with positiveswab results (n = 81 couples, prevalence ratio 1.7, 95% CI 1.1to 2.5) or male and female index subjects with positive PCRresults alone (n = 75 couples, prevalence ratio 1.7, 95% CI 1.2to 2.5) had no effect on our transmission estimates.

Non-persistence of NAAT-positive resultsIndex participants were screened initially in the emergencydepartment and re-evaluated at follow-up, thus it is possible toexamine the short-term persistence of NAAT-identified Ctrachomatis. Among participants who had not received antibiotictherapy during the emergency department visit and who hadboth NAAT and traditional assay results available for follow-up(n = 163; three participants had missing traditional assayresults), 27 (17%) individuals had no evidence of C trachomatisinfection by NAAT or culture/DFA. Nine (5%) individuals wereNAAT negative, but positive by culture/DFA. The remaining127 (78%) participants were NAAT positive (table 3).

In bivariable analyses, women were significantly more likelythan men to test negative for C trachomatis after a short follow-up(23% versus 6%, risk ratio 3.6, 95% CI 1.3 to 9.9) as wereindividuals who had used antibiotics in the three months beforetheir emergency department visit (32% versus 12%, risk ratio 2.6,95% CI 1.2 to 5.5). In multivariable analyses, only gender remainedsignificantly associated with the persistence of NAAT-positiveresults.

Non-persistence and partner concordanceWe also examined the association between the persistence ofNAAT-positive results and partnership concordance. Within the83 partnerships, only one partner of nine (11%) indexparticipants without evidence of C trachomatis infection atfollow-up tested positive. In contrast, 52 partners of 74 (70%)index participants with evidence of C trachomatis infection at

Figure 1 Subject participation in screening and follow-up, November2002 to February 2005. *Patients attending the emergency department(ED) were eligible for C trachomatis (CT) screening if they were between18 and 35 years of age, English-speaking, sexually active in the pastthree months, and a non-Hopkins employee or student. For the first fivemonths of data collection, respondents reporting antibiotic use within thepast three months were excluded. Patients were also ineligible if theywere critically ill or unable to participate as a result of a physicalcondition or cognitive impairment, or they had been previously enrolled inthe study and tested positive for C trachomatis. {A total of 700individuals did not consent, 82 completed the audio computer-assistedself interview only, 57 individuals were released from the emergencydepartment before completing the study, and 19 patients enrolled twice.{Subject consented and provided urine or self-administered swabspecimen for sexually transmitted infection testing using the nucleic acidamplification test (NAAT). 1As determined by repeatedly positive NAATon the same specimen. Initially male and female urine specimens weretested using ligase chain reaction (LCR) assay. After August 2003, maleurine and female self-administered vaginal swabs were tested using theCOBAS Amplicor assay. This change was necessitated by the specimenrequirements of the Roche Amplicor assay, which was used after AbbottLaboratories discontinued the LCR. "C trachomatis-positive indexsubjects who received antibiotic therapy during their emergencydepartment visit were not re-contacted for follow-up. **Forty subjectsprovided insufficient locating information, 44 received healthcareelsewhere, 40 received treatment only at the Johns Hopkins HospitalGeneral Clinical Research Center (GCRC), 16 did not show for theirscheduled GCRC appointment, and 22 either refused treatment, wereincarcerated, a non-resident of Baltimore, or in substance abuserehabilitation. {{After enrollment IDs were switched on two index

subjects’ specimens, two indexes were actually partners of twopreviously enrolled C trachomatis-positive index subjects, and onesubject did not have complete NAAT and traditional assay results.{{Seventeen partners were treated elsewhere, 20 were out of jurisdictionor not located, eight refused treatment, and five received treatment andan examination only at the GCRC. 11Two partners were enrolled at homeand provided specimens for NAAT only. An additional 14 partnershipswere missing complete NAAT and traditional assay results, one partnerwas enrolled six months after the index, and specimen IDs weremislabelled during collection for two partnerships.

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follow-up tested positive (prevalence ratio 6.3; 95% CI 0.98 to40.8).

DISCUSSIONNAAT provides enhanced sensitivity to detect chlamydialinfection. Understanding the clinical and public health implica-tions of the additional infections identified by NAAT is criticalfor the appropriate use of these tests. Using concordance as anestimate of transmission, we observed that individuals whowere positive by NAAT, but not by traditional assay, weresignificantly less likely than individuals who were positive byboth NAAT and traditional assay to have a concordantlyinfected sexual partner. Nonetheless, partner concordanceamong individuals with infections detected only by NAATwas moderate (45%). In addition, a substantial proportion ofindividuals (17–22%) who screened positive by NAAT for Ctrachomatis in the emergency department setting had noevidence of chlamydial infection after a short follow-up period(median three weeks).

A likely explanation for the diminished partner concordanceamong infections detectable only by NAAT is reduced organismburden. The enhanced sensitivity of NAAT increases thelikelihood of detecting infections with relatively few organisms.Alternatively, NAAT may detect the ‘‘passive presence’’ of theorganism after exposure, without a true, established infection.Consequently, individuals with infections detectable only byNAAT may be inherently less likely to transmit C trachomatis totheir partners.

We observed a surprisingly high incidence of infectionclearance, especially among women.10 This observation, coupledwith our findings regarding reduced concordance amongpartners of index participants whose follow-up NAAT wasnegative, suggests that at least some NAAT infections may becleared relatively rapidly, perhaps through antibiotic exposureor natural immune response,11 12 and not transmitted. Weobserved that many individuals without detectable infection atfollow-up had been treated with antibiotics in the three monthspreceding their initial screening, suggesting that NAAT may

Table 1 Characteristics of index subjects positive for C trachomatis who did and did not participate in follow-up

Characteristic

C trachomatis-positive indexparticipated in follow-up(N = 166)* N (%)

C trachomatis-positive indexdid not participate in follow-up(N = 172)* N (%) p{

Demographics

Age, in years, mean (SD) 22.5 (4.2) 23.5 (4.7) 0.04

Women 103 (62%) 87 (51%) 0.03

African American 137 (91%) 132 (88%) 0.26

Never married 132 (88%) 135 (89%) 0.82

Completed less than high school 60 (40%) 61 (40%) 0.98

Health behaviours

Previous C trachomatis or gonorrhoea infection 57 (38%) 61 (40%) 0.67

Dysuria and/or discharge past 3 months 48 (32%) 38 (25%) 0.20

Antibiotic use past 3 months 25 (17%) 29 (19%) 0.58

Condom use, past 5 sexual acts, mean (SD) 2.1 (2.0) 2.1 (2.0) 0.50

Illicit drug use past 30 days 79 (53%) 86 (57%) 0.54

Sexual behaviours

2+ Partners past 3 months 49 (33%) 62 (41%) 0.15

New partner past 3 months 55 (37%) 58 (40%) 0.72

Age of most recent partner, mean (SD) 24.2 (5.7) 25.4 (7.7) 0.12

Only heterosexual partners past 2 years 131 (90%) 130 (90%) 0.89

*11% of index patients did not complete emergency department interview.{Estimate for difference in measured characteristic between C trachomatis-positive index subjects who did and did not participate in follow-up, based on chi-square and t-tests forcategorical and continuous outcomes, respectively.

Table 2 Estimate of transmission, as defined by partner concordance, C trachomatis

Partner

Index

N+T+ (N = 52) N+T2 (N = 31) Total (N = 83)

Concordant

N+T+ 18 (35%) 5 (16%) 23 (28%)

N+T2 7 (13%) 5 (16%) 12 (14%)

N2T+ 14 (27%) 4 (13%) 18 (22%)

Total 39 (75%) 14 (45%) 53 (64%)

Discordant

N2T2 13 (25%) 17 (55%) 30 (36%)

Prevalence ratio 1.70 (95% CI 1.1 to 2.5)

N+, Nucleic acid amplification test (NAAT) positive; T+, traditional assay positive; T2, traditional assay negative.Concordant defined as NAAT and/or traditional assay positive for C trachomatis.Disconcordant defined as NAAT and traditional assay negative for C trachomatis.Tabulations include all partnerships with valid NAAT and traditional assay results: 72 index subjects with one partner, four indexeswith two partners, and one index with three partners. Fourteen index subjects with N2 results at follow-up (five N2T+ and nineN2T2) are considered N+ (based on their emergency department test result).Prevalence ratio and 95% CI represent the outcome of partner status of concordant versus discordant by index status and wereestimated from a generalised estimating equation logistic model that accounts for a lack of independence among index patientswith multiple partners.

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have detected residual DNA, rather than viable organisms, atthe initial screening evaluation.13 A proportion of the resultscould be false positives, although we re-tested all initial positiveresults to reduce this possibility. Sampling variability associatedwith the repeated testing of low-level infections is anotherpossibility, but appears less likely because of the multiplespecimens taken at follow-up.14 The likelihood of each of thesepossibilities is worthy of further clinical investigation.

The widespread application of NAAT has ‘‘resulted inconsiderable revision of our views of the clinical epidemiologyof C trachomatis’’.15 16 Only recently, however, have we begun toquestion the significance of some additional infections identifiedby NAAT.17 In this study, we examined partner concordance asa marker of the clinical significance of NAAT-identifiedinfections. Further study of the association between asympto-matic NAAT-identified chlamydial infections and other clinicalconsequences, eg the incidence of pelvic inflammatory disease,or inflammation, is needed.

Few studies have examined infection transmission withinpartnerships.18 We chose a non-sexually transmitted infectionclinic population as we were interested in understanding thetransmissibility of largely asymptomatic, unrecognised Ctrachomatis infections. Consequently, we screened over 6000individuals. In this cross-sectional sample, the timing of theprevalent infection in the index and the direction of transmis-sion between index and partner could not be established.Generalisation of our findings may be constrained by oursample selection and by incomplete recruitment, although indexparticipants had a similar risk profile to individuals who did notparticipate.

The advent of NAAT screening has expanded opportunitiesfor prevention of the serious consequences of untreated Ctrachomatis infection. Although we strongly believe thatindividuals with a positive NAAT should be informed and

treated, our study demonstrates that some NAAT-detectedinfections may not represent clinically active disease ortransmissible infections. Failure to diagnose a chlamydialinfection can negatively affect health, although incorrectlyidentifying individuals as infected can damage relationships.Patients, especially those with low-risk profiles or thosescreened from low C trachomatis prevalence populations,16

should be counselled about their test result accordingly.Guidelines for testing, patient counselling and managementshould be formulated so that users of this remarkable andpowerful tool—doctors, patients, and researchers—are aware ofthe advantages of NAAT and also its limitations.

Acknowledgements: The authors would like to thank Don Orr, Martina Morris, andHeather Miller for serving as scientific advisors to this project. They also thank SarahMobius for her managerial contribution to the study and Sheping Li at RTI forprogramming and data management; Ambreen Khalil and Chadd Krauss for theiroversight of interviews at the Johns Hopkins Adult Emergency Department; Joan Bess,Kenya S. Stewart and Nancy Willard of the Johns Hopkins Adolescent HealthResearch Group for outreach support; and Mary Ann Knott-Grasso, MS, CPNP, forproviding patient care at the Johns Hopkins General Clinical Research Center. Theauthors also wish to thank the laboratory personnel from the Department of Medicine,University of North Carolina at Chapel Hill, including Marcia Stedman, John Schmitz,and Dana Lapple; and Jeff Younger, Billie Jo Wood, and Hope L Johnson from theJohns Hopkins School of Medicine, Department of Infectious Diseases.

Funding: Primary support for this research was provided by National Institutes ofHealth (NIH) grant R01-HD039633 to SMR. RR was supported in part by a grant fromNCRR NIH 3M01RR00052-39-5(S1).

Competing interests: None.

Author contributions: SMR, WCM, CFT, PL, JE, RR and DK contributed to theconception and design of the study. SMR, WCM, CFT, JE, JZ, RR, CG and MHcontributed to the acquisition of data. SMR, WCM, MAV, AA-T and DK contributed toquestionnaire design. SMR, MAV and LG contributed to data management. SR, WCM,MAV and CFT contributed to the analysis and interpretation of data. All authorscontributed to writing the manuscript.Role of the funding source: The National Institutes of Health did not participate inthe design and conduct of the study, in the collection, analysis, and interpretation ofthe data, or in the preparation, review, or approval of the manuscript. Thecorresponding author had full access to all of the data in the study and takesresponsibility for the integrity of the data and the accuracy of the data analysis.

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Key messages

c Although the enhanced sensitivity of NAAT is well recognised,the significance and transmissibility of the additional infectionsdetected by NAAT are unknown.

c A substantial proportion of positive NAAT results forchlamydial infection may be of lower transmissibility and maynot persist after a short follow-up.

c Guidelines for testing, patient counselling and managementshould be formulated so that doctors, patients, andresearchers are aware of the advantages of NAAT as well asits limitations.

Table 3 Results of testing for C trachomatis at follow-up among NAAT-positive index participants

NAAT-positive index participants

Total Men Women

Follow-up test result

N2T2 27 (17%) 4 (6%) 23 (23%)

N2T+ 9 (5%) 3 (5%) 6 (6%)

N+T+ 83 (51%) 36 (57%) 47 (47%)

N+T2 44 (27%) 20 (32%) 24 (24%)

Total 163 63 100

Fisher’s exact p = 0.03

N+, Nucleic acid amplification test (NAAT) positive; T+, traditional assay positive; N2, NAAT negative; T2, traditional assaynegative.Fisher’s exact for test of association between gender and follow-up test result.

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10. Brunham RC, Plummer FA. A general model of sexually transmitted diseaseepidemiology and its implication for control. Med Clin North Am 1991;74:1339–52.

11. Moore SA, Sillekens PT, Jacobs MV, et al. Monitoring of Chlamydia trachomatisinfections after antibiotic treatment using RNA detection by nucleic acid sequencebased amplification. Mol Pathol 1998;51:149–54.

12. Bianchi A, Bogard M, Cessot G, et al. Kinetics of Chlamydia trachomatis clearance inpatients with azithromycin, as assessed by first void urine testing by PCR andtranscription-mediated amplification. Sex Trans Dis 1998;25:366–7.

13. Gaydos CA, Crotchfelt C, Howell MR, et al. Molecular amplification assays to detectchlamydial infections in urine specimens from high school female students and to monitorthe persistence of chlamydial DNA after therapy. J Infect Dis 1998;177:417–24.

14. Schachter J, Chow JM, Howard H, et al. Detection of Chlamydia trachomatis by nucleicacid amplification testing: our evaluation suggests that CDC-recommended approachesfor confirmatory testing are ill-advised. J Clin Microbiol 2006;44:2512–17.

15. Stamm WE. Chlamydia trachomatis—the persistent pathogen: Thomas ParranAward Lecture. Sex Transm Dis 2001;28:684–9.

16. Zenilman JM, Miller WC, Gaydos C, et al. LCR testing for gonorrhea and chlamydiain population surveys and other screenings of low prevalence populations: copingwith decreased positive predictive value. Sex Transm Infect 2003;79:94–7.

17. Hagdu A, Dendukuri N, Hilden J. Evaluation of a nucleic acid amplification test in theabsence of a gold-standard test: a review of the statistical and epidemiologicalissues. Epidemiology 2005;16:604–12.

18. Quinn T, Gaydos C, Shepherd M, et al. Epidemiologic and microbiologic correlates ofClamydia trachomatis infection in sexual partnerships. JAMA 1996;276:1737–42.

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Chlamydia

28 Sex Transm Infect 2008;84:23–28. doi:10.1136/sti.2007.027029

on 10 May 2008 sti.bmj.comDownloaded from

1

Concordance of Chlamydia trachomatis Infections Within Sexual Partnerships 1

2

Susan M. Rogers, PhD 3

William C. Miller, MD, PhD, MPH 4

Charles F. Turner, PhD 5

Jonathan Ellen, MD 6

Jonathan Zenilman, MD 7

Richard Rothman, MD, PhD 8

Maria Villarroel, MA 9

Alia Al-Tayyib, MSPH 10

Peter Leone, MD 11

Charlotte Gaydos, DrPH 12

Laxminarayana Ganapathi, PhD 13

Marcia Hobbs, PhD 14

David Kanouse, PhD 15

16

Author Affiliations: Program in Health and Behavior Measurement, Research Triangle 17

Institute, Washington, DC (Drs. Rogers, Turner, Ms Villarroel); Division of Infectious 18

Diseases, Department of Medicine and Epidemiology, University of North Carolina, 19

Chapel Hill (Drs Miller, Leone, Hobbs, Ms Al-Tayyib); City University of New York, 20

Queens College and Graduate Center (Dr Turner); Division of Adolescent Medicine, 21

School of Medicine, Johns Hopkins University (Dr Ellen); Division of Infectious Diseases, 22

School of Medicine, Johns Hopkins University (Drs Zenilman and Gaydos); Department 23

of Emergency Medicine, Johns Hopkins Medical Institution (Dr Rothman); Research 24

Computing Division, Research Triangle Institute, Research Triangle Park, NC (Dr 25

Ganapathi); RAND (Dr Kanouse). 26

2

Corresponding Author: Susan M. Rogers, Program in Health and Behavior 27

Measurement, Research Triangle Institute, 701 13th St NW, Suite 750, Washington, DC 28

20005 ([email protected]) 29

Funding/support: Primary support for this research was provided by NIH grant R01-30

HD039633 to Dr Rogers. Dr. Rothman was supported in part by a grant from NCRR NIH 31

3M01RR00052-39-5(S1). 32

3

Summary 33

Background Nucleic acid amplification tests (NAATs) offer new opportunities for 34

estimating the prevalence of untreated Chlamydia trachomatis infections (Ct), providing 35

substantial improvements in test sensitivity while maintaining the high specificity of 36

traditional testing methods. However, the transmissibility and public health significance 37

of some NAAT-identified infections are not known. We conducted a cross-sectional and 38

short-duration prospective cohort study with follow-up of participants with NAAT-39

identified chlamydial infection and their sexual partners to examine the potential 40

transmissibility and short-term persistence of NAAT-identified chlamydial infections. 41

Methods 6,094 adults aged 18 to 35 years attending an urban Emergency Department 42

(ED) from November 2002 through February 2005 provided specimens for Ct screening 43

using NAATs. Unrecognized Ct infections were identified in seven percent of ED 44

participants using NAAT. Participants testing positive were offered follow-up including 45

re-testing for Ct using NAAT and traditional methods, e.g. culture and direct fluorescent 46

antibody, and treated. Partners were offered identical evaluation and treatment 47

services. Overall, 90 Ct-positive participants had one or more sexual partners enrolled. 48

Results Evidence of transmission, as defined by concordance of infection between 49

sexual partners, was observed among 75% of partners of index cases testing positive by 50

both NAAT and traditional assay (N+T+) but only 45% of partners of index cases testing 51

positive by NAAT only (N+T-) (prevalence ratio 1.7, 95% CI 1.1, 2.5). Among index 52

participants returning for follow-up who had not received antibiotic therapy during the ED 53

visit, 17% had no evidence of Ct infection by NAAT or traditional assay (median follow-54

up = 3 weeks). 55

Interpretation A substantial proportion of positive NAAT results for chlamydial infection 56

may be of lower transmissibility and may not persist after a short follow-up. The public 57

health significance and long-term health effects of some positive NAATs are uncertain. 58

4

59

Introduction 60

Unrecognized and untreated C. trachomatis infection is common among young adults in 61

the United States. Nationwide, 4.7% of women and 3.7% of men aged 18 to 26 years 62

were estimated to have an untreated chlamydial (Ct) infection in 2001-2002.1 Among 63

adults aged 18 to 35 years in Baltimore, MD, untreated chlamydial infections were 64

detected in 6.4% of African American females; the majority of infections were 65

asymptomatic. 2 Given the significant potential morbidity associated with chlamydial 66

infection, including pelvic inflammatory disease and its consequences, controlling and 67

preventing undiagnosed C. trachomatis is a major public health concern. 3 68

69

Estimates of the prevalence of C. trachomatis at the population level have been 70

facilitated by the development of non-invasive nucleic acid amplification tests (NAATs) 71

that can use urine specimens or self-collected vaginal swabs. NAAT provides 72

substantial improvements in test sensitivity while retaining the high specificity of 73

traditional methods such as culture. The enhanced sensitivity of NAAT is due to an 74

extremely low limit of detection, with the potential to detect DNA (or RNA) of 75

approximately 10 organisms per milliliter of sample. 4 In contrast to culture, NAAT does 76

not require viable organisms. Given the high sensitivity and low limit of detection, the use 77

of NAAT, as compared to culture, increases the yield of infections detected by 20 to 40 78

percent. 5 79

80

Although the enhanced sensitivity of NAAT is well-recognized, the significance and 81

transmissibility of the additional infections detected by NAAT are unknown. If the 82

additional infections detected by NAAT are as transmissible as infections detected using 83

traditional methods, such as culture or DFA of culture transport media, the findings from 84

5

population studies call for new public health strategies to reduce infection rates. On the 85

other hand, it is possible that NAAT is detecting clinically inconsequential infections 86

involving extremely low levels of viable organisms, or perhaps amplifiable residual DNA 87

from a recently treated or controlled infection. In a previous population-based study, we 88

observed that many persons with NAAT-identified infection had few behavioral risk 89

factors and most were asymptomatic. 6 This raises the possibility that certain NAAT 90

infections may be of long duration and, presumably, low organism burden. Given the 91

extensive use of NAAT for the detection and diagnosis of chlamydial infection, a better 92

understanding of the clinical and public health significance of the additional infections 93

detected by NAAT is needed. 94

95

We conducted a cross-sectional study of adults attending an urban Emergency 96

Department and offered NAAT screening for chlamydial infection. We conducted follow-97

up of participants with untreated chlamydial infection and recruitment of their sexual 98

partners to explore the potential transmissibility of NAAT-identified infections. We 99

tested the hypothesis that chlamydial infections that are detectable only by NAAT are 100

less transmissible, as evidenced by infection concordance within partnerships, than 101

infections that also are detectable by traditional methods. In addition, a short-duration 102

prospective cohort study examined persistence of chlamydial infections among the 103

NAAT-positive participants who did and did not re-test Ct-positive (by NAAT and/or 104

traditional assay) when returning for follow-up treatment. 105

106

Methods 107

Study Population 108

Between November 2002 and February 2005, trained interviewers approached adult 109

patients attending the Johns Hopkins Hospital Emergency Department (JHH-ED) in 110

6

Baltimore, MD, USA, to assess eligibility for the study. We selected an ED population, 111

rather than a sexually transmitted infection (STI) clinic population, because our focus 112

was on largely asymptomatic and untreated NAAT-identified infections. Patients were 113

eligible for Ct screening if they were between 18 and 35 years of age, English-speaking, 114

and sexually active in the past 90 days. Employees and students of Johns Hopkins, and 115

patients who were critically ill (level-1 acute trauma patients), intoxicated, or presenting 116

for acute psychiatric or STI-related care were excluded. Initially, patients were not 117

eligible if they had used antibiotics within the past 30 days, but in April 2003 this 118

exclusion criterion was discontinued to enhance enrollment. Index participants enrolled 119

after March 2003 received a $10 food coupon for their participation. 120

121

We obtained contact information from all participants undergoing C. trachomatis testing 122

to facilitate notification and follow-up of positive test results by trained research Disease 123

Intervention Specialists (DIS). The DIS informed participants who did not receive 124

antibiotics during their ED visit of their positive result and offered follow-up examination, 125

additional Ct testing (NAAT and traditional assay), and treatment at the JHH General 126

Clinical Research Center (GCRC). Positive participants also were informed that they 127

could seek care from their private physician or the local health department. 128

129

After providing written informed consent, Ct-positive participants presenting to the GCRC 130

were asked to provide names of up to 5 sexual partners in the last 90 days. DIS 131

contacted named partners and offered evaluation and treatment procedures identical to 132

those provided to index participants. Index participants and partners were offered $50-133

200 in compensation for their time and travel costs. Participants who presented to the 134

GCRC but did not wish to complete the study were provided a free medical examination 135

and treatment. 136

7

137

The Research Triangle Institute, University of North Carolina, and Western (for The 138

Johns Hopkins University School of Medicine) Institutional Review Boards approved all 139

study procedures and modifications to the original protocol. Study participants with 140

positive test results for chlamydial infection were reported to the Baltimore City Health 141

Department. 142

143

Participant Interviews and Examinations 144

Participants in the ED completed a brief, approximately 8 minute, audio computer-145

assisted self interview (ACASI) about recent sexual and health behaviors.7 146

147

At follow-up, Ct-positive index participants presenting to the GCRC underwent physical 148

examination to detect clinical evidence consistent with chlamydial infection, including 149

visible discharge, genital ulcers, and lower abdominal and testicular tenderness. 150

Detailed self-reports of current and recent sex partners, partner-specific sexual 151

behaviors and STI history, use of antibiotics, STI symptoms, drug and alcohol use were 152

collected using a touch-screen ACASI. The follow-up interview took an average of 23 153

minutes to complete. 154

155

Sexual partners located by DIS staff were offered physical examinations, testing, and 156

treatment identical to index participants and asked to complete the ACASI. Partners 157

who did not attend the GCRC (and had not sought care elsewhere) were offered the 158

option of a home visit by a study DIS to complete the questionnaire and to collect a urine 159

or vaginal swab specimen for Ct testing. 160

161

Specimen Collection and Laboratory Testing 162

8

NAAT for Ct: FDA-approved NAAT was performed according to the manufacturers’ 163

instructions at the University of North Carolina at Chapel Hill. In the first nine months of 164

the study during ED screening, male participants provided a urine specimen and females 165

provided urine and self-administered vaginal swab specimens for Ct NAAT; however, 166

after August 2003, women provided only swabs. Initially, male and female urine 167

specimens were tested using a ligase chain reaction (LCR) assay (Abbott Laboratories, 168

North Chicago, Ill). After the LCR was withdrawn from the market in 2003 and the 169

laboratory’s supply of LCR kits was depleted, male urine specimens and female self-170

collected vaginal swabs swabs were tested using the COBAS Amplicor polymerase 171

chain reaction (PCR) assay (Roche Diagnostic Systems, Indianapolis, IN). Comparisons 172

of the performance characteristics of the NAATs suggest that the assays are similar for 173

the detection of chlamydial infection in urine and vaginal swab specimens. 8 9 Positive 174

NAAT results were confirmed by repeating the assay. Infection for C. trachomatis as 175

detected by NAAT (N+) was defined as a repeatedly positive test. 176

177

At follow-up, index participants with chlamydial infection and their sexual partners 178

provided specimens for multiple NAATs. Repeat NAAT was performed on urine (men 179

throughout the study and women during the initial study period), urethral swabs (men), 180

endocervical swabs (women), and self-administered vaginal swabs. Each index 181

participant thus received three NAATs: one upon initial recruitment in the ED and two 182

repeat tests on follow-up. Sexual partners received two NAATs during their GCRC clinic 183

visit. 184

185

Traditional assay for Ct: Traditional testing for C. trachomatis was performed at follow-186

up using culture and direct fluorescent antibody (DFA) test from urethral and 187

endocervical specimens in 2-sucrose phosphate culture transport media obtained from 188

9

male and female participants, respectively. Specimens were stored at -80C until testing 189

by the International STD Research Laboratory, Johns Hopkins University. Culture was 190

performed in McCoy cells. Culture-negative specimens were tested using DFA of the 191

sediment from the centrifuged culture transport media; a slide was considered DFA-192

positive if three or more elementary bodies were present. Culture-positive specimens 193

and DFA-positive/culture-negative specimens were considered positive for Ct by 194

traditional assay (T+). Specimens that were both culture and DFA negative were 195

considered traditional assay negative (T-). 196

197

Ct Genotyping 198

To determine the C. trachomatis serovar for organisms detected by NAAT, we amplified 199

variable portions of the ompA gene encoding the major outer-membrane protein 200

(MOMP) from a subset of NAAT-positive specimens using previously described primers 201

10 11 12 and sequenced the resulting PCR products on an ABI 3730 analyzer. We 202

compared sequences from clinical samples with ompA sequences of 17 C. trachomatis 203

serovars in the Gen-Bank database 13 and assigned the serovar of the best match to 204

each specimen. 205

206 207

Statistical Analyses and Outcomes 208

We used chi-square and t-tests to compare demographic and behavioral characteristics 209

of index participants with Ct-positive NAAT test results who did and did not present for 210

follow-up. 211

212

We assessed concordance of infection between sexual partners as a surrogate for 213

transmissibility of NAAT-identified chlamydial infections. We restricted this analysis to 214

10

partnerships in which both NAAT and traditional test results were available. We defined 215

concordance as either a positive NAAT (N+) or traditional assay (T+) result for C. 216

trachomatis among partners of Ct-positive index participants. All index participants were 217

considered NAAT-positive based on their ED test result. 218

219

Our primary hypothesis was that chlamydial infections detectable by NAAT but not by 220

traditional assay (N+T-) in the index participants would be less transmissible to sexual 221

partners than infections that also were detectable by traditional assay (N+T+). Non-222

concordance was considered as evidence of lower transmissibility. We tested this 223

hypothesis by examining prevalence ratios with 95% confidence intervals (CIs) that 224

compared the proportion of partnerships with concordant Ct infections (N+ and/or T+) 225

between index participants who tested N+T- and those that tested N+T+. 226

227

We also examined characteristics of index participants and their partners that may be 228

associated with infection concordance. These variables included age, number of recent 229

sexual partners, new partners within the past 90 days, history of chlamydial infection, 230

recent antibiotic use, reporting of symptoms, time to follow-up (days between index 231

screening in the ED and follow-up, days from index follow-up to partner follow-up, and 232

days between index screening and partner follow-up), and specimen type (female urine 233

vs vaginal swab, LCR vs PCR). 234

235

For all partnership analyses, we used generalized estimating equations (GEE) with a log 236

link and binomial error distribution to estimate prevalence ratios for concordance. GEE 237

account for within-group correlation, such as that which exists between multiple partners 238

of the same index participant.14 Additional analyses of partner concordance using only 239

11

single partnerships, e.g., the index and the first enrolled partner, yielded similar results 240

and are not presented. 241

242

As a secondary aim, we examined the persistence of NAAT-identified chlamydial 243

infections at follow-up among persons with positive NAAT in the ED. We defined 244

persistent infection as a positive NAAT in one or both specimens (urine or self-245

administered vaginal swab and clinician administered endocervical/urethral swab) or a 246

positive traditional test (culture or DFA) at follow-up. Factors potentially influencing 247

persistence of infection, including the number of days from initial screening to follow-up, 248

type of NAAT, respondent's gender, age, and previous diagnosis of chlamydial infection 249

(ever and within the past year) were examined in bivariable and multivariable binomial 250

regression analyses. In addition, we examined the association between persistence and 251

concordance of infection within partnerships. All statistical analyses were conducted 252

using Stata version 8 (Stata Corp., College Station, TX). 253

254

Role of the funding source 255

The US National Institutes of Health did not participate in the design and conduct of the 256

study, in the collection, analysis, and interpretation of the data, or in the preparation, 257

review, or approval of the manuscript. The corresponding author had full access to all of 258

the data in the study and takes responsibility for the integrity of the data and the 259

accuracy of the data analysis. 260

261

Results 262

Study Recruitment: Ct Screening and Follow-up 263

Over a 27-month period, 14,188 adult patients attending the JHH-ED were screened for 264

eligibility by 21 trained interviewers. Screening identified 6,952 English-speaking 265

12

sexually active 18-35 year old adults eligible for participation (Figure 1). Of these, 6,094 266

participants (87.7%) consented to chlamydial screening. 267

268

Overall, the prevalence of chlamydial infection was 7.0% in the ED participants. The 269

prevalence of Ct in male urine specimens (7%) was comparable as determined by LCR 270

and PCR (8.4% v 6.6%, p >0.10). Among female urine specimens tested by LCR 271

between November 2002 through August 2003, 6.8% were Ct positive; 7.3% of female 272

swab specimens collected during September 2003 through February 2005 tested 273

positive by PCR (p >0.10). 274

275

Of the 419 index participants who tested NAAT positive for chlamydial infection during 276

ED screening, 81 (19.3%) received antibiotic treatment during their ED visit and were not 277

eligible for follow-up. Of the remaining 338 eligible index participants, 166 (49%) 278

returned and enrolled in follow-up. Among those enrolled, the mean number of DIS 279

contacts was 2 (range, 1 to 10) and the average number of days between ED testing 280

and follow-up was 21.5 (range, 8 to 46 days). Participants who did and did not enroll in 281

follow-up were similar based on education, race/ethnicity, marital status, previous STI, 282

and recent sexual behaviors (Table 1). In comparison to participants who did not enroll, 283

participants enrolled in follow-up were slightly younger (mean age, 22.5 vs 23.5 years; p 284

= 0.04) and more were female (62% vs 50.6%, p = 0.03). 285

286

Most index participants enrolled at follow-up (87%) named one or more recent sexual 287

partners; 22 (13%) refused to provide partner information. Of the 175 partners identified, 288

152 (86.9%) were contacted successfully by the DIS and 102 (58.3%) were enrolled. 289

One hundred partners were examined and treated; two partners refused evaluation and 290

were interviewed at home. Overall, 90 Ct-positive index participants had one or more 291

13

sexual partners enrolled; 79 index participants had one partner, 10 indexes had two 292

partners, and 1 index had three partners enrolled. Nearly one-half (48%) of the 293

partnerships enrolled in the study presented for follow-up together. 294

295

Partner Concordance of Ct Infections 296

Our analyses of Ct partnerships are limited to the 83 heterosexual couples (72 index 297

participants with one partner, four indexes with two partners, and one index with three 298

partners) for whom both NAAT and traditional assay results were available. We 299

excluded 17 partnerships because of missing NAAT or traditional assay results for the 300

index subject or their partner(s), inadequate specimen collection, transcription error, or 301

multiple enrollment of a positive index subject. In addition, two exclusively male 302

partnerships were omitted from these analyses as we did not collect anal or throat 303

specimens. 304

305

Evidence of transmission of chlamydial infection, as defined by concordance within 306

partnerships, was more common among index cases testing positive by both NAAT and 307

traditional assay (N+T+) than among index cases testing positive by NAAT only (N+T-) 308

(Table 2). Evidence of transmission was observed in 39 of 52 partners (75%) of N+T+ 309

index cases, but only 14 of 31 partners (45%) of N+T- index cases (prevalence ratio 1.7, 310

95% CI: 1.1, 2.5). 311

312

The relationship between test result of the index (N+T- or N+T+) and partner 313

concordance did not vary by gender. Among male partners of N+T+ female indexes, 314

82% were concordant for Ct, compared to 46% of male partners of N+T- females. 315

Similarly, 70% of female partners of N+T+ index males were concordant, in comparison 316

to 43% of female partners of males testing positive by NAAT only. There was no 317

14

difference by the index’s or partner’s age, number of new partners in the past 3 months, 318

history of chlamydial infection, history of any STI, or antibiotic use prior to the follow-up 319

visit. Adjusting for time from screening of the index to follow-up visit also had no 320

substantial effect. However, adjusting for the timing of the partner visit reduced the 321

prevalence ratio to 1.5 (95% CI: 1.03, 2.2). Partners presenting at the same time as the 322

index participant were the most likely to test concordant. Partners presenting after the 323

index participants were less likely to demonstrate concordance (1 – 7 days: prevalence 324

ratio 0.62; 95% CI: 0.41, 0.93; > 7 days: prevalence ratio 0.65; 95% CI: 0.39, 1.07; 325

referent = 0 days). 326

327

Limiting traditional test results to culture only decreased our overall estimate of 328

concordance from 64% to 54% (prevalence ratio = 1.4, 95% CI 0.93, 2.0). It is possible 329

that differences in NAAT results by type of NAAT (LCR vs PCR) or specimen type for 330

females (urine vs vaginal swab) could influence our transmission estimates. Although all 331

women screened in the ED provided vaginal swabs for Ct testing using PCR, initially 332

women provided urine specimens, in addition to swabs, for testing by LCR. When the 333

LCR was withdrawn in 2003, male urines and female swabs were tested using PCR. 334

Restricting our concordance analysis to include: 1) only female indexes with positive 335

swab results (n=81 couples, prevalence ratio 1.7, 95% CI 1.1, 2.5) or 2) male and female 336

index subjects with positive PCR results alone (n=75 couples, prevalence ratio 1.7, 95% 337

CI 1.2, 2.5) had no effect on our transmission estimates. 338

339

Non-persistence of NAAT Positive Results 340

Index participants were screened initially in the ED and re-evaluated at follow-up; thus it 341

is possible to examine the short-term persistence of NAAT-identified Ct. Among 342

participants who had not received antibiotic therapy during the ED visit and who had 343

15

both NAAT and traditional assay results available for follow-up (n=163; 3 participants 344

had missing traditional assay results), 27 (17%) persons had no evidence of Ct infection 345

by NAAT or culture/DFA. Nine (5%) persons were NAAT-negative, but positive by 346

culture/DFA; the remaining 127 (78%) participants were NAAT-positive (Table 3). 347

348

In bivariable analyses, women were significantly more likely than men to test negative 349

for Ct after a short follow-up (23% versus 6%, risk ratio 3.6, 95% CI: 1.3, 9.9). Persons 350

who had used antibiotics in the 3 months prior to their visit in the ED were also more 351

likely to test negative at follow-up (32% versus 12%, risk ratio 2.6, 95% CI: 1.2, 5.5). 352

Index participant’s age, previous chlamydial infection, type of NAAT, and time between 353

ED testing and the follow-up visit were not associated with infection status at follow-up. 354

In multivariable analyses, only gender remained significantly associated with persistence 355

of NAAT-positive results. 356

357

Non-persistence and Partner Concordance 358

We also examined the association between persistence of NAAT-positive results and 359

concordance of infection within partnerships. Within the 83 partnerships, only one 360

partner of 9 (11%) index participants without evidence of Ct infection at follow-up tested 361

positive. In contrast, 52 partners of 74 (70%) index participants with evidence of Ct 362

infection at follow-up tested positive for Ct (prevalence ratio 6.3; 95% CI: 0.98, 40.8). 363

364

C. trachomatis typing 365

To determine whether the nucleic acids amplified by NAAT in concordant partnerships 366

represented infection by the same chlamydial strain, we compared C. trachomatis 367

serovars established from the DNA sequences of variable portions of the ompA gene, 368

encoding the major outer-membrane protein amplified in specimens from NAAT-positive 369

16

concordant couples. In 19 of 21 couples with identifiable serovars, chlamydial 370

genotypes matched exactly. Serovar D was the most commonly identified (30%), 371

followed by Ia (25%), F (20%), and E (10%). Serovars J (5%), Ja (5%), and K (5%) 372

each were identified in one couple. 373

374

Discussion 375

NAAT technology provides enhanced sensitivity to detect chlamydial infection and 376

increased opportunities for chlamydial screening compared to traditional testing 377

methods. Understanding the clinical and public health implications of the additional 378

infections identified by NAAT is critical for appropriate use of these tests. Using 379

concordance as an estimate of transmission, we observed that persons who were 380

positive by NAAT, but not by traditional assay, were significantly less likely than persons 381

who were positive by both NAAT and traditional assay to have a concordantly infected 382

sexual partner. Nonetheless, partner concordance among persons with infections 383

detected only by NAAT was moderate (45%). In addition, a substantial proportion of 384

persons (17%-22%) who screened positive by NAAT for Ct in the ED setting had no 385

evidence of chlamydial infection by NAAT and/or traditional assay after a short follow-up 386

period (median = 3 weeks). 387

388

These results suggest that while many C. trachomatis infections detected by NAAT 389

persist and are transmissible within sexual partnerships, the significance of some 390

infections detected by these tests is unclear. This uncertainty derives from the same 391

factors that are responsible for the advantages of the assay – a low limit of detection and 392

the ability to detect DNA without viable organisms. 393

394

17

A likely explanation for the diminished partner concordance among infections detectable 395

only by NAAT is reduced organism burden. The enhanced sensitivity of NAAT increases 396

the likelihood of detecting infections with relatively few organisms. Alternatively, NAAT 397

may detect ‘passive presence’ of the organism after exposure, without a true, 398

established infection. Consequently, persons with infections detectable only by NAAT 399

may be inherently less likely to transmit Ct to their partners. 400

401

The probability of transmission is influenced by factors other than organism burden, such 402

as frequency of intercourse and previous exposure. Unfortunately, because of 403

ambiguities in partner specification for participants with multiple partners, we did not 404

have precise quantitative information on the frequency of intercourse within partnerships. 405

In our study, previous self-reported chlamydial infection did not influence the relationship 406

between test result of the index and partner concordance. 407

408

We observed a surprisingly high incidence of infection clearance, especially among 409

women. Our observed clearance rate (7.8 cases per 1000 person-days) is consistent 410

with an estimated average duration of infection of 128 days, considerably shorter than 411

the commonly cited 365 days.15 This observation, coupled with our findings regarding 412

reduced concordance among partners of index participants whose follow-up NAAT was 413

negative, suggests that at least some NAAT-identified infections may be cleared 414

relatively rapidly and not transmitted. Some of these may be infections that are close to 415

being resolved, whether through antibiotic exposure or natural immune response. 16,17 416

We observed that many persons without detectable infection at follow-up had been 417

treated with antibiotics in the 3 months preceding their initial screening, suggesting that 418

NAAT may have detected residual DNA, rather than viable organisms, at the initial 419

screening evaluation 18. A proportion of the screening results could be false positives, 420

18

although we re-tested initial positive results to reduce this possibility. Sampling variability 421

associated with repeated testing of individuals with low-level infections 19 is another 422

possibility, but appears less likely because of the multiple specimens taken at follow-up. 423

The likelikhood of each of these possibilities is not known but is worthy of further clinical 424

investigation. 425

426

Since their widespread introduction more than a decade ago, the use of NAAT has 427

“resulted in considerable revision of our views of the clinical epidemiology of C. 428

trachomatis”. 20 Not only has NAAT suggested an increased prevalence of infection in 429

nearly every population tested, but it has also allowed expansion of screening programs 430

to non-clinical settings, thereby enhancing screening services available to men and to 431

asymptomatic individuals. 21 Only recently, however, have we begun to question the 432

clinical and public health significance of some additional infections identified by NAAT. 433

11,22 In this study, we examined partner concordance as a marker of the clinical 434

significance of NAAT-identified infections. Further study of the association between 435

asymptomatic NAAT-identified chlamydial infections and other clinical consequences, 436

e.g., incidence of PID, inflammation, is needed. 437

438

Very few studies have examined infection transmission within partnerships 23. Not only 439

are such studies logistically and technically challenging, but they are expensive to 440

conduct. Our study design was intended to enhance our understanding of the likelihood 441

of transmission of NAAT-diagnosed infection in a sexual partnership – and to do so with 442

minimal risk to our study subjects. Several alternate study designs were considered by 443

our research team, but ultimately rejected. For ethical reasons, we chose not to test 444

index subjects for chlamydia using traditional assay. Only subjects who tested positive 445

for chlamydia by NAAT and their recent sexual partners had urethral or cervical samples 446

19

obtained for culture during the clinical examination. We chose an Emergency 447

Department population rather than a STI clinic setting as we were interested 448

understanding the clinical consequences of largely asymptomatic and unrecognized Ct 449

infections detectable by NAAT but not by traditional methods. As a result, it was 450

necessary to screen over 6,000 individuals. We used concordance as an estimate of 451

transmission. Because we began with a cross-sectional screening evaluation, the timing 452

of the prevalent infection in the index case was unknown. Consequently, the direction of 453

transmission between index and partner could not be established. Generalization of our 454

findings may be constrained by our sample selection – patients recruited from an urban 455

Emergency Department – and by the incomplete recruitment of index participants and 456

their partners. In general, index participants had a similar risk profile to persons who 457

were not recruited into the study. We were unable to compare characteristics of 458

partners who did and did not enroll; however, it seems unlikely that characteristics on 459

which selection might occur would bias enrollment with respect to traditional test status, 460

the basis for our primary hypothesis. 461

462

The advent of NAAT screening for chlamydial infection has vastly expanded 463

opportunities for prevention of the serious consequences of untreated infection. 464

However, questions remain regarding the clinical and public health consequences of 465

some infections detected by NAAT. The interpretation of a positive NAAT or any other 466

screening method as an ipso facto indicator of disease can have important personal 467

implications. While failure to diagnose a chlamydial infection can negatively affect 468

health, incorrectly identifying individuals as infected can damage relationships. Because 469

many Ct infections identified by NAAT screening are asymptomatic and easily treated, 470

there has been a tendency to presume that a positive result indicates the presence of 471

disease, with the responsibility to inform and provide treatment. While we strongly 472

20

believe that persons with a positive NAAT should be informed and treated, our study 473

demonstrates that some NAAT-detected infections may not represent clinically active 474

disease or transmissible infections. Patients, especially those with low risk profiles or 475

those screened from a low Ct-prevalence population, 21 should be counseled about their 476

test result accordingly. Guidelines for testing, patient counseling and management 477

should be formulated so that users of this remarkable and powerful tool -- physicians, 478

patients, and researchers -- are aware of not only NAAT’s advantages, but also its 479

limitations. 480

481 482

Contributors: S Rogers, W Miller, C Turner, P Leone, J Ellen, R Rothman, and D 483

Kanouse contributed to the conception and design of the study. S Rogers, W Miller, C 484

Turner, J Ellen, J Zenilman, R Rothman, C Gaydos, M Hobbs contributed to the 485

acquisition of data. S Rogers, W Miller, M Villarroel, A Al-Tayyib, and D Kanouse 486

contributed to questionnaire design. S Rogers, M Villarroel, and L Ganapathi 487

contributed to data management. S Rogers, W Miller, M Villarroel and C Turner 488

contributed to the analysis and interpretation of data. All authors contributed to writing 489

the manuscript. 490

Conflicts of Interest: We declare that we have no conflicts of interest. 491

Acknowledgment: We thank Don Orr, Martina Morris, and Heather Miller for serving as 492

scientific advisors to this project. We also thank Sarah Mobius for her managerial 493

contribution to the study and Sheping Li at RTI for programming and data management; 494

Ambreen Khalil and Chadd Krauss for their oversight of interviews at the Johns Hopkins 495

Adult Emergency Department; Joan Bess, Kenya S. Stewart and Nancy Willard of the 496

Johns Hopkins Adolescent Health Research Group for outreach support; and Mary Ann 497

Knott-Grasso, MS, CPNP for providing patient care at the Johns Hopkins General 498

21

Clinical Research Center. We also thank the laboratory personnel from the Department 499

of Medicine, University of North Carolina at Chapel Hill, including Marcia Stedman, John 500

Schmitz, and Dana Lapple; and Jeff Younger, Billie Jo Wood, and Hope L. Johnson from 501

the Johns Hopkins School of Medicine, Department of Infectious Diseases. 502

503

504

1 Miller WC, Ford CA, Morris M, et al. Prevalence of chlamydial and gonococcal infections among young adults in the United States. JAMA 2004; 291(18):2229-36. 2 Turner CF, Rogers SM, Miller HG, et al. Untreated gonococcal and chlamydial infection in a probability sample of adults. JAMA 2002; 287(6):726-33. 3 Institute of Medicine. The Hidden Epidemic: Confronting Sexually Transmitted Diseases. Washington, DC: National Academy Press, 1996. 4 Schacter J. Chlamydia trachomatis: the more you look the more you find – how much is there? Sex Transm Dis 1998; 25(5): 229-31. 5 Stamm WE. Chlamydia trachomatis infections of the adult. In: Holmes KK, Sparling PF, Mardh P, Lemon SM, Samm WE, Piot P, Wasserheit JN, eds. Sexually Transmitted Diseases. New York: McGraw Hill, 1999, 407-22. 6 Rogers SM, Miller HG, Miller WC, Zenilman JC, Turner CF. NAAT-identified and self-reported gonorrhea and chlamydial infections: different at-risk population subgroups? Sex Transm Dis 2002; 29(10):588-96. 7 Cooley PC, Rogers SM, Turner CF et al. Using touch-screen audio-CASI to obtain data on sensitive topics. Comp in Human Beh 2001; 17:285-93. 8 Watson EJ, Templeton A, Russell I, Paavonen J, Mardh PA, Stary A, Pederson BS. The accuracy and efficacy of screening tests for Chlamydia trachomatis: A systematic review. J Med Microbiol 2003; 51:1021-1031. 9 Schacter J, McCormack, Chernesky M, Martin DH, Van Der Pol B, Rice PA, Hook EW, Stamm WE, Quinn TC, Chow JM. Vaginal swabs are appropriate specimens for diagnosis of genital tract infection with Chlamydia trachomatis. 2003. J of Clin Microbiol 41(8): 3784-3789. 10 Bandea CI, Kubota K, Brown TM, et al. Typing of Chlamydia trachomatis strains from urine samples by amplification and sequencing the major outer membrane protein gene (omp1). Sex Transm Infect 2001; 77:419-422. 11 Lysen M, Osterlund A, Rubin CJ, et al. Characterization of ompA genotypes by sequence analysis of DNA from all detected cases of Chlamydia trachomatis infections

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during 1 year of contact tracing in a Swedish County. J Clin Microbiol 2004; 42:1641-1647. 12 Dean D, Stephens RS. Identification of individual genotypes of Chlamydia trachomatis from experimentally mixed serovars and mixed infections among trachoma patients. J Clin Microbiol 1994; 32:1506-1510). 13 Gen-Bank Accession numbers AF118868, AY535166, DQ064279, DQ064289, DQ064291, DQ064292, DQ064293, DQ064299, M14738, M17342, M17343, M36533, U78528, X52080, X55700, X62919, X62921. 14 Hardin JW, Hilbe JM. Generalized Estimating Equations. Boca Raton, Fl: Chapman and Hall/CRC, 2003. 15 Brunham RC, Plummer FA. A general model of sexually transmitted disease epidemiology and its implication for control. Med Clin North Am 1991; 74(6):1339-52. 16 Moore SA, Sillekens PT, Jacobs MV et al. Monitoring of Chlamydia trachomatis infections after antibiotic treatment using RNA detection by nucleic acid sequence based amplification. Mol Pathol 1998; 51(3):149-54. 17 Bianchi A, Bogard M, Cessot G et al. Kinetics of Chlamydia trachomatis clearance in patients with azithromycin, as assessed by first void urine testing by PCR and transcription-mediated amplification. Sex Trans Dis 1998; 25(7):366-7. 18 Gaydos CA, Crotchfelt C, Howell MR, Kralian S, Hauptman P, Quinn TC. Molecular amplification assays to detect chlamydial infections in urine specimens from high school female students and to monitor the persistence of chlamydial DNA after therapy. Journal of Infectious Diseases 1998; 177(2):417-424. 19 Schachter J, Chow JM, Howard H, Bolan G, and Moncada J. Detection of Chlamydia trachomatis by nucleic acid amplification testing: Our evaluation suggests that CDC-recommended approaches for confirmatory testing are ill-advised. J Clin Micro 2006; 44(7):2512-17. 20 Stamm WE. Chlamydia trachomatis—The persistent pathogen: Thomas Parran Award Lecture. Sex Transm Dis 2001; 28(12):684-89. 21 Zenilman JM, Miller WC, Gaydos C, Rogers SM, Turner CF. LCR Testing for gonorrhea and chlamydia in population surveys and other screenings of low prevalence populations: Coping with decreased positive predictive value. Sex Transm Infect 2003; 79(2):94-7. 22 Hagdu A, Dendukuri N, Hilden J. Evaluation of a nucleic acid amplification test in the absence of a gold-standard test: a review of the statistical and epidemiological issues. Epidemiology 2005; 16(5):604-12. 23 Quinn T, Gaydos C, Shepherd M, Bobo L, Hook EW, Viscidi R, Rompalo A. Epidemiologic and microbiologic correlates of chlamydia trachomatis infection in sexual partnerships. Journal of the American Medical Association 1996; 276(21):1737-1742.

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1

Figure 1. Subject participation in screening and follow-up, November 2002-February 2005

CT Screening of Index Subjects

14,188 ED patients approached

6,952 (49%) eligible for participation a

858 ineligible b

6,094 (87.7%) received NAAT screening c

86 specimens unprocessed

419 (7.0%) CT+ d

81 received antibiotic therapy in ED e

Follow-up and Evaluation of CT+ Subjects

338 CT+ index subjects contacted for follow-up

172 did not participate f

166 CT+ index subjects participated in follow-up g

Partner Evaluation

175 partners identified

152 partners contacted by Disease Intervention Specialists

50 partners did not participate h

102 partners of CT+ index subjects participated in follow-up

19 partnerships excluded i

83 partnerships valid NAAT and traditional assay results

Notes: a Patients attending the Emergency Department were eligible for Ct screening if they were between 18 and 35 years of age, English-speaking, sexually active in the past 3 months, and a non-Hopkins employee or student. For the first 5 months of data collection, respondents reporting antibiotic use within the past 3 months were excluded. Patients also were ineligible if: they were critically ill or unable to participate due to a physical condition or cognitive impairment, or they had been previously enrolled in the study and tested positive for CT. b 700 subjects did not consent, 82 completed the ACASI only, 57subjects were released from the ED prior to completing the study, and 19 patients enrolled twice. c Subject consented and provided urine or self-administered swab specimen for STI testing using NAAT.

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d As determined by repeatedly positive NAAT on the same specimen. Initially male and female urine specimens were tested using LCR assay (Abbott Laboratories). After August 2003, male urine and female self-administered vaginal swabs were tested using the COBAS Amplicor assay (Roche Diagnostics). This change was necessitated by the specimen requirements of the Roche Amplicor assay which was used after Abbott Laboratories discontinued the LCR. e Ct+ index subjects who received antibiotic therapy during their ED visit were not re-contacted for follow-up. f 40 subjects provided insufficient locating information, 44 received healthcare elsewhere, 40 received treatment only at the GCRC, 16 did not show for their scheduled GCRC appointment, and 22 either refused treatment, were incarcerated, a non-resident of Baltimore, or in substance abuse rehabilitation. g Following enrollment, IDs were switched on 2 index subjects' specimens, 2 indexes were actually partners of 2 previously enrolled Ct-positive index subjects, and 1 subject did not have complete NAAT and traditional assay results. h 17 partners were treated elsewhere, 20 were out of jurisdiction or not located, 8 refused treatment, and 5 received treatment and an examination only at the GCRC. i Two partners were enrolled at home and provided specimens for NAAT only. An additional 14 partnerships were missing complete NAAT and traditional assay results, 1 partner was enrolled 6 months after the index, and specimen IDs were mislabeled during collection for 2 partnerships.

3

Table 1. Characteristics of index subjects positive for C. trachomatis who did and did not participate in follow-up

Characteristic

CT+ Index participated in

Follow-up (N=166) a

CT+ Index did not participate in

Follow-up (N=172) a p b

N (%) N (%)

Demographics

Age, in years (mean ± SD) 22.5±4.2 23.5±4.7 0.04

Female 103 (62%) 87 (51%) 0.03

African American 137 (91%) 132 (88%) 0.26

Never married 132 (88%) 135 (89%) 0.82

Completed less than high school 60 (40%) 61 (40%) 0.98

Health behaviors

Prior CT or GC infection 57 (38%) 61 (40%) 0.67

Dysuria and/or discharge past 3 mos 48 (32%) 38 (25%) 0.20

Antibiotic use past 3 mos 25 (17%) 29 (19%) 0.58

Condom use, past 5 sexual acts (mean ± SD) 2.1±2.0 2.1±2.0 0.50

Illicit drug use past 30 days 79 (53%) 86 (57%) 0.54

Sexual behaviors

2+ partners past 3 mos 49 (33%) 62 (41%) 0.15

New partner past 3 mos 55 (37%) 58 (40%) 0.72

Age of most recent partner (mean ± SD) 24.2±5.7 25.4±7.7 0.12

Only heterosexual partners past 2 years 131 (90%) 130 (90%) 0.89

Notes: a 11% of index patients did not complete ED interview. b Estimate for difference in measured characteristic between CT+ index subjects who did and did not participate in follow-up, based on chi-square and t-tests for categorical and continuous outcomes, respectively.

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Table 2. Estimate of transmission, as defined by partner concordance, Chlamydia trachomatis Index N+T+ N+T- Total Partner (N=52) (N=31) (N=83)

Concordant N+T+ 18 (35%) 5 (16%) 23 (28%) N+T- 7 (13%) 5 (16%) 12 (14%) N-T+ 14 (27%) 4 (13%) 18 (22%)

Total 39 (75%) 14 (45%) 53 (64%)

Discordant N-T- 13 (25%) 17 (55%) 30 (36%)

Prevalence ratio: 1.70 (95% CI, 1.1-2.5) Notes: N+, NAAT positive; T+, traditional assay positive; T-, traditional assay negative Concordant defined as NAAT and/or traditional assay positive for C. trachomatis. Disconcordant defined as NAAT and traditional assay negative for C. trachomatis. Tabulations include all partnerships with valid NAAT and traditional assay results: 72 index subjects with 1 partner, 4 indexes with 2 partners, and 1 index with 3 partners. Fourteen index subjects with N- results at followup (5 N-T+ and 9 N-T-) are considered N+ (based on their ED test result). Prevalence ratio and 95% CI represent the outcome of partner status of concordant versus discordant by index status and were estimated from GEE logistic model that accounts for lack of independence among index patients with multiple partners.

5

Table 3. Results of testing for C. trachomatis at follow-up among NAAT positive index participants

NAAT positive index participants

Total Male Female Follow-up test result

N-T- 27 (17%) 4 ( 6%) 23 (23%) N-T+ 9 ( 5%) 3 ( 5%) 6 ( 6%) N+T+ 83 (51%) 36 (57%) 47 (47%) N+T- 44 (27%) 20 (32%) 24 (24%) Total 163 63 100

Fisher’s exact p=0.03

Notes: N+, NAAT positive; T+ traditional assay positive; N- NAAT negative; T- traditional assay negative